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• W2899557002 abstract "A chiral lipidomics approach was established for comprehensive profiling of regio- and stereoisomeric monoepoxy and monohydroxy metabolites of long-chain PUFAs as generated enzymatically by cytochromes P450 (CYPs), lipoxygenases (LOXs), and cyclooxygenases (COXs) and, in part, also unspecific oxidations. The method relies on reversed-phase chiral-LC coupled with ESI/MS/MS. Applications revealed partially opposing enantioselectivities of soluble and microsomal epoxide hydrolases (mEHs). Ablation of the soluble epoxide hydrolase (sEH) gene resulted in specific alterations in the enantiomeric composition of endogenous monoepoxy metabolites. For example, the (R,S)/(S,R)-ratio of circulating 14,15-EET changed from 2.1:1 in WT to 9.7:1 in the sEH-KO mice. Studies with liver microsomes suggested that CYP/mEH interactions play a primary role in determining the enantiomeric composition of monoepoxy metabolites during their generation and release from the ER. Analysis of human plasma showed significant enantiomeric excess with several monoepoxy metabolites. Monohydroxy metabolites were generally present as racemates; however, Ca2+-ionophore stimulation of whole blood samples resulted in enantioselective increases of LOX-derived metabolites (12S-HETE and 17S-hydroxydocosahexaenoic acid) and COX-derived metabolites (11R-HETE). Our chiral approach may provide novel opportunities for investigating the role of bioactive lipid mediators that generally exert their physiological functions in a highly regio- and stereospecific manner. A chiral lipidomics approach was established for comprehensive profiling of regio- and stereoisomeric monoepoxy and monohydroxy metabolites of long-chain PUFAs as generated enzymatically by cytochromes P450 (CYPs), lipoxygenases (LOXs), and cyclooxygenases (COXs) and, in part, also unspecific oxidations. The method relies on reversed-phase chiral-LC coupled with ESI/MS/MS. Applications revealed partially opposing enantioselectivities of soluble and microsomal epoxide hydrolases (mEHs). Ablation of the soluble epoxide hydrolase (sEH) gene resulted in specific alterations in the enantiomeric composition of endogenous monoepoxy metabolites. For example, the (R,S)/(S,R)-ratio of circulating 14,15-EET changed from 2.1:1 in WT to 9.7:1 in the sEH-KO mice. Studies with liver microsomes suggested that CYP/mEH interactions play a primary role in determining the enantiomeric composition of monoepoxy metabolites during their generation and release from the ER. Analysis of human plasma showed significant enantiomeric excess with several monoepoxy metabolites. Monohydroxy metabolites were generally present as racemates; however, Ca2+-ionophore stimulation of whole blood samples resulted in enantioselective increases of LOX-derived metabolites (12S-HETE and 17S-hydroxydocosahexaenoic acid) and COX-derived metabolites (11R-HETE). Our chiral approach may provide novel opportunities for investigating the role of bioactive lipid mediators that generally exert their physiological functions in a highly regio- and stereospecific manner. A wide array of bioactive lipid mediators is generated through oxygenation reactions from PUFAs, such as arachidonic acid (AA), EPA, and DHA. Oxygenated PUFAs have also been termed “oxylipins” and comprise metabolites formed by cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P450 (CYP) enzymes as well as nonenzymatic oxidation reactions (1Smilowitz J.T. Zivkovic A.M. Wan Y.J. Watkins S.M. Nording M.L. Hammock B.D. German J.B. Nutritional lipidomics: molecular metabolism, analytics, and diagnostics.Mol. Nutr. Food Res. 2013; 57: 1319-1335Crossref PubMed Scopus (44) Google Scholar, 2Gabbs M. Leng S. Devassy J.G. Monirujjaman M. Aukema H.M. Advances in our understanding of oxylipins derived from dietary PUFAs.Adv. Nutr. 2015; 6: 513-540Crossref PubMed Scopus (397) Google Scholar, 3Willenberg I. Ostermann A.I. Schebb N.H. Targeted metabolomics of the arachidonic acid cascade: current state and challenges of LC-MS analysis of oxylipins.Anal. Bioanal. Chem. 2015; 407: 2675-2683Crossref PubMed Scopus (77) Google Scholar, 4Schunck W.H. Konkel A. Fischer R. Weylandt K.H. Therapeutic potential of omega-3 fatty acid-derived epoxyeicosanoids in cardiovascular and inflammatory diseases.Pharmacol. Ther. 2018; 183: 177-204Crossref PubMed Scopus (119) Google Scholar). Current methods of targeted lipidomics allow high-throughput, comprehensive, and highly sensitive quantitation of oxylipins in biological and clinical samples (5Astarita G. Kendall A.C. Dennis E.A. Nicolaou A. Targeted lipidomic strategies for oxygenated metabolites of polyunsaturated fatty acids.Biochim. Biophys. Acta. 2015; 1851: 456-468Crossref PubMed Scopus (104) Google Scholar). Most of these analytical approaches rely on LC coupled with MS/MS. Thereby, LC is performed on achiral stationary phases under reversed-phase conditions and MS mostly uses ESI for efficient ionization of the analytes. These advanced methods can measure more than one hundred different oxylipin species in one analytical run, but are unable to distinguish between the enantiomers (5Astarita G. Kendall A.C. Dennis E.A. Nicolaou A. Targeted lipidomic strategies for oxygenated metabolites of polyunsaturated fatty acids.Biochim. Biophys. Acta. 2015; 1851: 456-468Crossref PubMed Scopus (104) Google Scholar). The lack of enantiomeric resolution is an inherent property of the achiral-LC-ESI-MS/MS approaches. This feature limits the conclusions that can be drawn regarding the enzymatic versus nonenzymatic origin of oxylipin species or the biological significance of changes in the endogenous oxylipin profile, e.g., in the course of cardiovascular and inflammatory diseases. To address these questions, different strategies of targeted chiral lipidomics are under investigation (6Mesaros C. Blair I.A. Targeted chiral analysis of bioactive arachidonic acid metabolites using liquid-chromatography-mass spectrometry.Metabolites. 2012; 2: 337-365Crossref PubMed Scopus (28) Google Scholar). Chiral-LC has been primarily developed for normal-phase conditions using apolar solvent systems that preclude ESI application for efficient ionization. A way out of this problem is provided by electron capture atmospheric pressure chemical ionization (ECAPCI)-MS. This method can be coupled with normal-phase chiral-LC and requires prior derivatization of oxylipins, e.g., into their pentafluorobenzyl esters (6Mesaros C. Blair I.A. Targeted chiral analysis of bioactive arachidonic acid metabolites using liquid-chromatography-mass spectrometry.Metabolites. 2012; 2: 337-365Crossref PubMed Scopus (28) Google Scholar). Using this sensitive approach, a recent study showed stereospecific responses to in vitro stimulation of human blood samples regarding the formation of HETEs (7Mazaleuskaya L.L. Salamatipour A. Sarantopoulou D. Weng L. FitzGerald G.A. Blair I.A. Mesaros C. Analysis of HETEs in human whole blood by chiral UHPLC-ECAPCI/HRMS.J. Lipid Res. 2018; 59: 564-575Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). An alternative strategy takes advantage of chiral stationary phases that are compatible with water-containing polar mobile phases. This approach allows performing reversed-phase chiral-LC coupled with ESI-MS/MS detection and quantitation of the resolved stereoisomers. Reversed-phase chiral-LC-ESI-MS/MS has been successfully used for the determination of enantiomers as well as diastereomeric epimers of pro-resolving lipid mediators (8Oh S.F. Vickery T.W. Serhan C.N. Chiral lipidomics of E-series resolvins: aspirin and the biosynthesis of novel mediators.Biochim. Biophys. Acta. 2011; 1811: 737-747Crossref PubMed Scopus (43) Google Scholar, 9Homann J. Lehmann C. Kahnt A.S. Steinhilber D. Parnham M.J. Geisslinger G. Ferreiros N. Chiral chromatography-tandem mass spectrometry applied to the determination of pro-resolving lipid mediators.J. Chromatogr. A. 2014; 1360: 150-163Crossref PubMed Scopus (14) Google Scholar, 10Toewe A. Balas L. Durand T. Geisslinger G. Ferreiros N. Simultaneous determination of PUFA-derived pro-resolving metabolites and pathway markers using chiral chromatography and tandem mass spectrometry.Anal. Chim. Acta. 2018; 1031: 185-194Crossref PubMed Scopus (13) Google Scholar). The present study was aimed at developing an analytical system suitable for comprehensive profiling of regio- and enantiomeric monoepoxy and monohydroxy metabolites in biological samples. The metabolites to be targeted included epoxyeicosatrienoic acids (EETs) and HETEs derived from AA, epoxyeicosatetraenoic acids (EEQs) and hydroxyeicosapentaenoic acids (HEPEs) derived from EPA, and epoxydocosapentaenoic acids (EDPs) and hydroxydocosahexaenoic acids (HDHAs) derived from DHA. While each of the monoepoxy metabolite regioisomers can be formed as (R,S)- or (S,R)-enantiomers, the hydroxyl group in the various regioisomeric monohydroxy metabolites can exist in R- or S-configuration. After optimizing the set-up and conditions for reversed-phase chiral-LC-ESI-MS/MS, we tested the applicability of the established procedure first by analyzing the enantioselectivities of soluble epoxide hydrolases (sEHs) and microsomal epoxide hydrolases (mEHs) in metabolizing CYP-derived EETs, EEQs, and EDPs. Subsequent studies served to elucidate the profile of the targeted oxylipin stereoisomers in plasma and liver samples from WT and sEH-KO mice, as well as in human plasma. The enantiomers of 8,9-, 11,12-, and 14,15-EET (11Zhang J.Y. Blair I.A. Direct resolution of epoxyeicosatrienoic acid enantiomers by chiral-phase high-performance liquid chromatography.J. Chromatogr. B Biomed. Appl. 1994; 657: 23-29Crossref PubMed Scopus (13) Google Scholar), as well as of 17,18-EEQ and 19,20-EDP (12Arnold C. Markovic M. Blossey K. Wallukat G. Fischer R. Dechend R. Konkel A. von Schacky C. Luft F.C. Muller D.N. et al.Arachidonic acid-metabolizing cytochrome P450 enzymes are targets of omega-3 fatty acids.J. Biol. Chem. 2010; 285: 32720-32733Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar) were prepared as described previously by resolving the racemic mixtures (±) of the respective monoepoxides, as purchased from Cayman Chemicals, using chiral-LC on Chiralcel OD or OB columns (Daicel, Illkirch, France). Authentic samples of 17(R),18(S)- and 17(S),18(R)-EEQ were produced using recombinant CYP1A1 and CYP102, respectively (13Schwarz D. Kisselev P. Ericksen S.S. Szklarz G.D. Chernogolov A. Honeck H. Schunck W.H. Roots I. Arachidonic and eicosapentaenoic acid metabolism by human CYP1A1: highly stereoselective formation of 17(R),18(S)-epoxyeicosatetraenoic acid.Biochem. Pharmacol. 2004; 67: 1445-1457Crossref PubMed Scopus (117) Google Scholar). It was assumed that the 19,20-EDP enantiomers elute in the same order as the 17,18-EEQ enantiomers; however, authentic standards were not available to verify their identities. The (R,S) and (S,R) enantiomers collected from chiral-LC were quantitated using achiral-LC-MS/MS as described below. All racemic oxylipins as well as the currently available set of R- and S-enantiomers of HETEs, HEPEs, and HDHAs were purchased from Cayman Chemicals. If authentic stereoisomers were not available for a given oxylipin, steric configurations were not assigned; instead, they were designated as peak 1 (P1) and P2, according to the order of their elution from chiral-LC. The internal standard (IS) solution was prepared in acetonitrile and contained 0.5 #x03BC;g/ml each of 15(S)-HETE-d8 and ±8,9-EET-d11. Bond Elut Certify II columns (3 ml, 200 mg sorbent) for solid phase extraction (SPE) were purchased from Agilent Technologies. Acetonitrile, methanol, glacial acetic acid, water (LC-MS grade), and buffers were purchased from VWR International. N-[1-(1-oxopropyl)-4-piperidinyl]N′-[4-(trifluoromethoxy)phenyl]urea (TPPU) was bought from Cayman Chemicals. Cyclohexene oxide (CHO), BSA (fatty acid free), and ketamine/xylazine K-113 were purchased from Sigma-Aldrich and Na-heparin from Braun Melsungen. Columns containing chiral polysaccharide-based stationary phases were purchased from Phenomenex (Aschaffenburg, Germany). For the #x201C;chiral-1#x201D; method (compare Fig. 1), Lux-Cellulose-3 (150 × 2 mm, 3 #x03BC;m particles) was coupled upstream with an Agilent ZORBAX Eclipse Plus C18 column (50 × 2.1 mm, 1.8 #x03BC;m particles). Mobile phase was a linear gradient of methanol/water/glacial acetic acid from 70:30:0.05 to 80:20:0.05 (v/v/v) over 16 min at a flow rate of 0.4 ml/min, followed by washing with 100#x0025; methanol over 4 min. For the #x201C;chiral-2#x201D; method, Lux-Amylose-1 (150 × 2 mm, 3 #x03BC;m particles) was coupled upstream with an Agilent ZORBAX SB-C8 column (50 × 2.1 mm, 1.8 #x03BC;m particles). Mobile phase was a linear gradient of acetonitrile/methanol/water/glacial acetic acid from 27:3:70:0.05 to 63:7:30:0.05 (v/v/v/v) over 30 min at a flow rate of 0.4 ml/min. Before use, the chiral column systems were carefully adjusted to appropriate pressure (pressure maximum set at 300 bar) by increasing the flow rate over 10 min from 0 to 0.4 ml/min, followed by an equilibration period of 1 h. Retention times of analytes became stable after two runs, as tested using a mix of authentic standard compounds for quality control (QC). Routinely, an equilibration time of 15 min between successive analytical runs was used. Before long-term storage, the chiral columns were washed with acid-free 95#x0025; organic mobile phase. For the achiral system, an Agilent Zorbax Eclipse Plus C18 column (150 × 2.1 mm, 1.8 #x03BC;m particles) was used as stationary phase. Chromatography was done under gradient conditions at a flow rate of 0.3 ml/min starting with acetonitrile/water/acetic acid in a ratio of 5:95:0.05 (v/v/v) for 0.5 min, followed by increases to 56:44:0.05 within 5 min, to 61:39:0.05 within 5 min, and finally to 87:13:0.05 within 13 min, followed by washing the column with 98:2:0.05 for 6.5 min. All columns were kept at 40°C and the autosampler at 6°C. The injection volume was 10 #x03BC;l (15 #x03BC;l for achiral analysis). LC-MS/MS analysis was performed using an Agilent 6490 Triple Quad mass spectrometer (Agilent Technologies, Santa Clara, CA) coupled with an UHPLC system of the Agilent 1290 Infinity series. ESI operated in the negative ionization mode. The instrument parameters and conditions for multiple reaction monitoring (MRM) are given in supplemental Table S1. Peak detection, integration, and quantitation were done using Agilent MassHunter software. 15(S)-HETE-d8 and ±8,9-EET-d11 served as ISs for quantifying monohydroxides and monoepoxides, respectively. Ten-point-calibration curves were obtained based on analyte/IS peak area ratios after injecting 10 #x03BC;l of stock solutions in acetonitrile that contained 0–25 ng of the analytes and 5 ng IS per 100 #x03BC;l. Limit of detection was defined as the lowest concentration that gave a signal-to-noise (S/N) ratio >3. Lower limit of quantitation (LLOQ) was defined as the lowest concentration that gave a S/N ratio >10. The S/N ratio was calculated using the adjacent area corresponding to one peak width of the analyte as reference. Accuracy and precision were tested analyzing QC samples. The QC samples were used at concentrations corresponding to the low, mid, and upper parts of the linear calibration curves and were measured three times. #x201C;Accuracy I#x201D; represents intra-day deviations and was expressed as percent difference of the measured mean from the expected concentration as calculated from the respective calibration function. Precision was defined as the relative SD (RSD) of the three repeated measures. #x201C;Accuracy II#x201D; of the chiral methods was expressed as percent difference between the mean sum measured for a given pair of enantiomers and the corresponding total metabolite level as measured by the achiral method. Deuterated standards were purchased from Cayman Chemicals [±8,9-EET-d11, ±11,12-EET-d11, ±14,15-EET-d11, 5(S)-HETE-d8, 12(S)-HETE-d8, and 15(S)-HETE-d8]. A mix of these standards (5 ng each) was used to spike extracts (n = 6 per group) prepared as described below from 30 mg murine liver tissue or 0.5 ml human plasma reconstituted in 100 #x03BC;l of acetonitrile/water (60:40). Identically spiked solvent samples served as control. Matrix effects were calculated as percentage of the peak areas measured by chiral-LC-MS/MS after adding the standards to the extracts compared with the pure solvent. Recombinant human sEH was purchased from Cayman Chemicals and diluted in incubation buffer containing 0.1#x0025; fatty acid-free BSA. Pooled human liver microsomes were purchased from Thermo Fisher Scientific. Murine liver microsomal and cytosolic fractions were prepared as described previously (14Muller D.N. Schmidt C. Barbosa-Sicard E. Wellner M. Gross V. Hercule H. Markovic M. Honeck H. Luft F.C. Schunck W.H. Mouse Cyp4a isoforms: enzymatic properties, gender- and strain-specific expression, and role in renal 20-hydroxyeicosatetraenoic acid formation.Biochem. J. 2007; 403: 109-118Crossref PubMed Scopus (135) Google Scholar). The hydrolase assays were performed at 30°C in 300 #x03BC;l of Tris-HCl buffer (50 mM) with 0.01#x0025; fatty acid-free BSA, pH 7.5. Substrates were used at a final concentration of 10 #x03BC;M. To determine sEH-mediated hydrolyses, the assays were started by adding the indicated amounts of human sEH or liver cytosolic fraction prepared from WT mice. mEH activities were determined using human liver or murine liver microsomal protein (prepared from sEH-KO mice). Incubations with human liver microsomes were performed in the presence of 2 #x03BC;M TPPU. The reactions were stopped by adding 15 #x03BC;l of 400 mM citric acid and 1 ml of ethyl acetate. The ethyl acetate extracts were then evaporated, dissolved in acetonitrile/water (60:40), and analyzed using chiral-LC-MS/MS. Incubations for analyzing liver microsomal AA metabolism were performed at 37°C in a total volume of 100 #x03BC;l of 100 mM potassium phosphate buffer (pH 7.2) containing 80 #x03BC;g of the microsomal protein and the substrate at a final concentration of 10 #x03BC;M. After preincubating the microsomes with AA for 5 min, reactions were started with NADPH (1 mM final concentration) and terminated after 10 min by adding 5 #x03BC;l of 0.4 M citric acid. In control experiments, NADPH was omitted. To exclude potentially contaminating sEH activities, murine liver microsomes were prepared from sEH-KO mice and the reactions with human liver microsomes were performed in the presence of the sEH inhibitor, TPPU (2 #x03BC;M). CHO was used to inhibit mEH activities (15Magdalou J. Hammock B.D. 1,2-Epoxycycloalkanes: substrates and inhibitors of microsomal and cytosolic epoxide hydrolases in mouse liver.Biochem. Pharmacol. 1988; 37: 2717-2722Crossref PubMed Scopus (21) Google Scholar, 16Morisseau C. Newman J.W. Dowdy D.L. Goodrow M.H. Hammock B.D. Inhibition of microsomal epoxide hydrolases by ureas, amides, and amines.Chem. Res. Toxicol. 2001; 14: 409-415Crossref PubMed Scopus (52) Google Scholar); the final CHO concentrations were 250 #x03BC;M with murine or 800 #x03BC;M with human liver microsomes. Incubations containing 0.1#x0025; ethanol were performed as vehicle controls. Reaction products were extracted into ethyl acetate, evaporated under nitrogen, dissolved in acetonitrile/water (60:40), and analyzed using chiral-LC-MS/MS. Plasma samples (0.3–0.5 ml) or 10–20 mg aliquots of homogenized liver tissue mixed with 0.5 ml of distilled water containing 4 #x03BC;M TPPU, were filled up with 1.5 ml of acetonitrile containing 50 #x03BC;g of butylated hydroxytoluene and the IS [5 ng each of 15(S)-HETE-d8 and ±8,9-EET-d11]. Next, 0.3 ml of 10 M sodium hydroxide were added and the samples were incubated for 30 min at 60°C. After alkaline hydrolysis, 0.3 ml of acetic acid/water (58:42, v/v) and 3.5 ml of phosphate buffer (Sorensen's buffer, pH 6.0, containing 5#x0025; methanol) were added. The pH was controlled and, if necessary, adjusted to pH 6.0. The samples were then cleared by centrifugation and extracted by SPE. The SPE columns were preconditioned with 3 ml of methanol, followed by 6 ml of Sorensen's buffer, pH 6.0, containing 5#x0025; methanol. Columns were loaded with the samples and washed with 3 ml of methanol/water (1:1, v/v). Metabolites and IS were eluted with 2 ml of hexane/ethyl acetate (75:25, v/v) containing 1#x0025; acetic acid. The eluates were transferred into vials preloaded with 20 #x03BC;l of glycerol/methanol (1:9, v/v) and then evaporated to dryness on a heating block at 40°C under a stream of nitrogen. Residues were dissolved in 100 #x03BC;l of acetonitrile/water (60:40) and either stored at −80°C or immediately applied to LC-MS/MS. All animal experiments were performed in accordance with the guidelines of the Charité University Berlin and approved by local German authorities (LaGeSo, G0146/16, Berlin, Germany). WT and sEH-KO mice were originally established by Boehringer-Ingelheim Pharmaceuticals and were then further backcrossed for nine generations onto C57BL/6ByJ before being used in our studies (17Monti J. Fischer J. Paskas S. Heinig M. Schulz H. Gosele C. Heuser A. Fischer R. Schmidt C. Schirdewan A. et al.Soluble epoxide hydrolase is a susceptibility factor for heart failure in a rat model of human disease.Nat. Genet. 2008; 40: 529-537Crossref PubMed Scopus (150) Google Scholar, 18Zhu Y. Blum M. Hoff U. Wesser T. Fechner M. Westphal C. Gurgen D. Catar R.A. Philippe A. Wu K. et al.Renal ischemia/reperfusion injury in soluble epoxide hydrolase-deficient mice.PLoS One. 2016; 11: e0145645PubMed Google Scholar). The animals were kept under specific pathogen-free conditions with a standard 12:12 h light-dark cycle and had ad libitum access to water and standard chow. Starting with an age of 10–13 weeks, male WT and sEH-KO mice (n = 6 per group) received a diet enriched in EPA and DHA over a period of 3 weeks. The diet (EF R/M, containing 5#x0025; sunflower and 2.5#x0025; Omacor oil from SNIFF Spezialitäten GmbH, Soest, Germany) was composed as described in detail previously (12Arnold C. Markovic M. Blossey K. Wallukat G. Fischer R. Dechend R. Konkel A. von Schacky C. Luft F.C. Muller D.N. et al.Arachidonic acid-metabolizing cytochrome P450 enzymes are targets of omega-3 fatty acids.J. Biol. Chem. 2010; 285: 32720-32733Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Mice were anesthetized with ketamine/xylazine (240 mg/kg) containing heparin (220 IU/kg). Blood (0.5–1.0 ml) was collected and plasma was prepared immediately by centrifugation (10,000 g, 10 min). Plasma as well as harvested liver and other organ samples were snap-frozen in liquid nitrogen and stored at −80°C. Before analysis, organs were homogenized in liquid nitrogen using a BioPulverizer (BioSpec Products Inc., USA). The human blood samples analyzed in the present study originated from a previous trial, where we treated 20 healthy volunteers with an EPA/DHA supplement and analyzed concomitant changes in the formation of AA-, EPA-, and DHA-derived metabolites (19Fischer R. Konkel A. Mehling H. Blossey K. Gapelyuk A. Wessel N. von Schacky C. Dechend R. Muller D.N. Rothe M. et al.Dietary omega-3 fatty acids modulate the eicosanoid profile in man primarily via the CYP-epoxygenase pathway.J. Lipid Res. 2014; 55: 1150-1164Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The blood samples used here for chiral-LC-MS/MS analysis were withdrawn after the participants received dietary EPA/DHA supplementation for 8 weeks. One set of the corresponding plasma samples (n = 6; three male and three female) was immediately prepared by centrifugation. Another set of plasma samples was prepared after in vitro incubation of fresh whole blood samples (4.5 ml) with the Ca2+-ionophore, A23187 (50 #x03BC;M), or its vehicle (1#x0025; DMSO) for 30 min at 37°C (n = 6 per group). Samples were stored at −80°C until analysis. Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Inc., La Jolla, CA). All tests were done with the nonparametric Mann-Whitney U test. A probability value of P < 0.05 was considered significant. Data are shown as mean ± SEM for the animal experiments and as mean ± SD for the in vitro experiments. Figure 1 shows the principal setup of the analytical procedure developed for comprehensive profiling of regio- and stereoisomeric monoepoxy and monohydroxy metabolites in biological samples. Using aliquots of the extracted metabolites, chiral-LC was performed on two different stationary phases, each coupled with ESI-MS/MS detection. The chiral-1 and chiral-2 methods resolved and enabled measurement of distinct subsets of enantiomeric metabolites, comprising most of the targeted monoepoxides on Cellulose-3 (Table 1) and all monohydroxides as well as several mid-chain monoepoxides on Amylose-1 (Table 2), respectively. A third aliquot was used to perform reversed-phase LC on a C18 column followed by ESI-MS/MS detection. This #x201C;achiral#x201D; method was included to obtain reference data on the total levels of all targeted metabolites, independent of their enantiomeric composition.TABLE 1.Chromatographic parameters of the chiral-1 methodCompoundLLOQ (ng)aNanograms absolute.Linear Range (ng)aNanograms absolute.R2Retention Time (RSD)bRSD (#x0025;) related to nine QC samples at three different concentrations.Resolution RcChromatographic resolution of the enantiomeric pair as calculated by R = 1.18 · [(tR2 − tR1)/(FWHM1 + FWHM2)] where tR is retention time (minutes) and FWHM is full peak width at half maximum (minutes); formula according to (20). (RSD)bRSD (#x0025;) related to nine QC samples at three different concentrations.8(R),9(S)-EET0.250.25–250.998813.02 (0.3)2.0 (3.4)8(S),9(R)-EET0.250.25–250.998913.93 (0.2)14(R),15(S)-EET0.100.1–250.998512.98 (0.2)1.6 (5.7)14(S),15(R)-EET0.100.1–250.998413.68 (0.2)8,9-EEQ peak 10.100.05–250.999412.23 (0.3)2.5 (9.4)8,9-EEQ peak 20.100.05–250.997513.23 (0.4)14,15-EEQ peak 1<0.050.05–250.998611.54 (0.3)2.2 (2.9)14,15-EEQ peak 2<0.050.05–250.997712.41 (0.2)17(R),18(S)-EEQ<0.050.1–250.998611.20 (0.2)1.5 (4.3)17(S),18(R)-EEQ<0.050.1–250.998511.78 (0.2)16,17-EDP peak 10.250.1–250.998614.69 (0.2)2.7 (4.9)16,17-EDP peak 20.250.1–250.998715.89 (0.4)19(R),20(S)-EDP0.250.25–250.998914.68 (0.3)2.2 (5.4)19(S),20(R)-EDP0.250.25–250.999115.65 (0.3)a Nanograms absolute.b RSD (#x0025;) related to nine QC samples at three different concentrations.c Chromatographic resolution of the enantiomeric pair as calculated by R = 1.18 · [(tR2 − tR1)/(FWHM1 + FWHM2)] where tR is retention time (minutes) and FWHM is full peak width at half maximum (minutes); formula according to (20Ding J. Zhang M. Dai H. Lin C. Enantioseparation of chiral mandelic acid derivatives by supercritical fluid chromatography.Chirality. 2018; Crossref Scopus (9) Google Scholar). Open table in a new tab TABLE 2.Chromatographic parameters of the chiral-2 methodCompoundLLOQ (ng)aNanograms absolute.Linear Range (ng)aNanograms absolute.R2Retention Time (RSD)bRSD (#x0025;) related to nine QC samples at three different concentrations.Resolution RcR = 1.18 · [(tR2 − tR1)/(FWHM1 + FWHM2)] where tR is retention time (minutes) and FWHM is full peak width at half maximum (minutes); formula according to (20). (RSD)5(R)-HETE<0.050.05–100.990922.03 (0.0)5.7 (6.6)5(S)-HETE<0.050.987223.47 (0.0)9(R)-HETE0.100.25–250.999321.79 (0.1)3.6 (10.7)9(S)-HETE0.100.994420.63 (0.1)11(R)-HETE<0.050.25–100.997421.51 (0.1)6.4 (4.7)11(S)-HETE<0.050.999523.02 (0.1)12(R)-HETE<0.050.05–100.987122.87 (0.0)5.4 (3.3)12(S)-HETE<0.050.991124.43 (0.1)15(R)-HETE0.250.05–100.987222.02 (0.1)8.8 (5.3)15(S)-HETE0.250.990924.54 (0.1)19(R)-HETE0.250.25–100.990619.57 (0.1)0.6 (18.5) dBaseline separation of the corresponding enantiomers was not reached (R < 1.5); however, reliable validation experiments (see supplemental Table S3) and quantitation in biological matrices were still possible.19(S)-HETE0.250.989919.41 (0.1)5(R)-HEPE<0.050.05–100.99719.51 (0.1)4.4 (4.0)5(S)-HEPE<0.050.990720.59 (0.1)9(R)-HEPE<0.050.05–100.993319.19 (0.0)3.9 (4.6)9(S)-HEPE<0.050.993920.11 (0.1)12(R)-HEPE0.250.25–250.993120.18 (0.1)5.7 (14.8)12(S)-HEPE0.100.989321.61 (0.1)15(R)-HEPE<0.050.05–100.992919.38 (0.0)4.9 (5.1)15(S)-HEPE<0.050.991620.60 (0.1)18-HEPE peak 1<0.050.05–100.992518.43 (0.1)3.0 (2.2)18-HEPE peak 2<0.050.992919.14 (0.1)17(R)-HDHA<0.050.25–100.999321.85 (0.1)4.9 (9.1)17(S)-HDHA<0.050.993822.97 (0.1)11(R),12(S)-EET<0.050.5–100.997926.77 (0.0)1.5 (7.7)11(S),12(R)-EET<0.050.99426.37 (0.1)11,12-EEQ peak 1<0.050.25–250.999423.55 (0.0)0.7 (10.1) dBaseline separation of the corresponding enantiomers was not reached (R < 1.5); however, reliable validation experiments (see supplemental Table S3) and quantitation in biological matrices were still possible.11,12-EEQ peak 2<0.050.999223.76 (0.1)7,8-EDP peak 1<0.050.25–100.997526.18 (0.0)3.6 (5.0)7,8-EDP peak 2<0.050.99727.18 (0.1)13,14-EDP peak 1<0.050.25–100.997925.98 (0.0)0.7 (9.6) dBaseline separation of the corresponding enantiomers was not reached (R < 1.5); however, reliable validation experiments (see supplemental Table S3) and quantitation in biological matrices were still possible.13,14-EDP peak 2<0.050.998426.20 (0.1)a Nanograms absolute.b RSD (#x0025;) related to nine QC samples at three different concentrations.c R = 1.18 · [(tR2 − tR1)/(FWHM1 + FWHM2)] where tR is retention time (minutes) and FWHM is full peak width at half maximum (minutes); formula according to (20Ding J. Zhang M. Dai H. Lin C. Enantioseparation of chiral mandelic acid derivatives by supercritical fluid chromatography.Chirality. 2018; Crossref Scopus (9) Google Scholar).d Baseline separation of the corresponding enantiomers was not reached (R < 1.5); however, reliable validation experiments (se" @default.
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• W2899557002 title "Chiral lipidomics of monoepoxy and monohydroxy metabolites derived from long-chain polyunsaturated fatty acids" @default.
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